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59
CHAPTER 3
SPECTROPHOTOMETRIC DETERMINATION OF AMMONIA IN INORGANIC
COMPOUNDS
3.1 . Introduction
Nitrogen constitutes 78.09% by volume of dry
unpolluted air, but it is a minor element with respect
to its abundance in the earthj:"crust. Atmospheric nitrogen
forms an important raw material for the economical
synthesis of bulk chemicals of industrial importance,
viz. ammonia, urea, nitrate, nitrite etc. Therefore,
estimation of ammonia and its derivatives assumes a great
importance for chemical industry. Traditionally..,ammonia
has been estimated by direct titration with an acid.
Kjeldahl method based on the conversion of nitrogen
containing organic compounds to ammonium ion, is in use
since 1883. The procedure is time proven for the
determination of nitrogen in wide variety of chemical
compounds. The various methods for the analysis of
ammonia include titrimetric, spectrophotometric and
chromatographic technique.
3.1.1 Titrimetric Determination of Ammon~
(i) Acid-base titration
Ammonia is titrated directly with standard acid
solution using methyl red as an indicator.85
The end
60
point of the titration is also determined potentio
metrically. 86 Ammonium ion in salts is titrated with
standard sodium hydroxide solution conductometri-
11 87-89 ca y.
Ammonia is treated with an excess of acid, which
is then back titrated with standard alkali solution. 90
Ammonium ion reacts with the mercury-EDTA complex. f
The hydrogen ion formed is titrated with standard alkali
1 t. 91 so u ion.
(ii) Distillation Methods
( The ammonium salt is treated with sodium hydroxide, ~
followed by distillation of liberated ammonia into
standard solution of hydrochloric or sulphuric acid and
the excess of acid is back titrated with standard alkali
1 t. 92 so u ion. Only volatile basic substances such as
pyridine and other amines interfer in the determination.
Alternatively, the liberated ammonia on treatment
with alkali is absorbed in boric acid solution or in
nickel ammonium sulphate solution. In the first case,
the ammonium borate formed is titrated directly against
standard acid solution using a mixture of methyl red-
methylene blue as an indicator. In the second case,
61
nickel-amine complex formed is titrated against a
standard acid solution. Both these methods require
only one standerd solution.93
The ammonium salts are analysed by
treatment of the sample directly with excess sodium
hydroxide solution. The solution is boiled to remove
ammonia, and the excess of alkali is titrated with f
t d d .d l t' 92 Th th d . l lt s an ar aci so u ion. e me o gives ow resu s,
mainly due to loss of alkali by spray and attack by hot
alkali on the glass apparatus.
(iii) Formaldehyde Met!!£2,__
Ammonium salts react with formaldehyde to give
hexamethylenetetramine and an acid. The later is titrated
against standard alkali using phenolphthalein as an
. d. t 93 in ice or.
(iv) ~ohalide Oxidation Procedures
Ammonium salts are determined by their decomposition
with sodium hypobromite to nitrogen gas. However, the
reaction is not considered to be quantitative.94
Therefore,,
a correction factor should be used in the determination.
2NH3
+ 3Na0Br =
62
Addition of highly alkaline sodium hypobromite solution
is found to give better results. The use of standard
solution of potassium bromate as a source of bromine
instead of potassium bromide is reported to give
quantitative results. The'unreacted hypobromite is
determined iodometrically. 95
Arcand and5wift96
utiliz;d the hypobromite
reaction as the basis of a coulometric titration •
• Bromine generated by the oxida+.ion of bromide ion at
platinum anode, is converted to hypobromite. The method
has been useful to determine ammonia in low concen-
trations.
The use of calcium hypochlorite solution to
generate hypobromite ion in situ by oxidation of pota
ssium bromide has been exhaustively studied by Kolthoff
and Stenger.97 In the titration of ammonia in the
presence of hypochlorite, the end point is detected by
the reaction of bromine with Bordeaux dye. The
determination is also carried out by adding excess of
hypochlorite and the unreacted reagent is titrated
with standard sodium thiosulphate solution. In another
method, excess of hypochlorite is reduced with excess
63
sodium arsenite and the residual reducing agent is
titrated with sodium hypochlorite solution. The above
reactions are carried out in presence of bicarbonate
or in alkaline medium. The pH of the reaction medium
is critical. The loss of ammonia by, volatilization and
by partial oxidation to nitrous oxide increases with
increase in pH. The optimum pH for the reaction is
B.2 to B.5.
Laitinen and Woerner98 • 99 have titrated ammonia
with hypochlorite in a bicarbonate medium containing
bromide amperometrically.
A coulometric procedure is described by Liberti
and Lazzari using hypohalite.100
3.1.2 Gravimetric Methods
There are no specific quantitative gravimetric
methods for the determination of ammonium ions. Ammonium
ion is estimated as chloroplatinate.101
However,this
method is not specific as almost all ions except chlo-
~ideinterfer in the procedure.
Ammonium tetraphenylborate obtained by the reaction
of ammonium ion with tetraphenyl borate, is determined
. t . 11 1 02 gravime rica y.
~-
64
The thermogravimetric pyrolysis of the ammonium
salt has been studied.103
It was found that ammonium
tetraphenylborate sublimes at 130°c. This observation
forms the basis of quantitative determination of ammonia.
3.1.3 ~eectroehotometric Method
Several colorimetric methods have been reported 1·
for the determination of ammonia or ammonium ions.
Nessler's end Indophenol method are the most widely used
procedures for determination of ammonia. Both the
procedures are describe in detail in Chapter 4. Other
methods are summarized briefly below :
Ammoni~ reacts with cupric ion to form cupric
ammonium complex which is measured at 700 nm.104
Ammonia
is oxidized to nitrogen by hypobromite and excess of the
hypobro~ite is determined by measuring absorbance at
330 nm. 105
Ammonia reacts with bispyrazolone in the presence of
hl . T t f. . k . 1 t 1 d b. · "d 106 • 1 D7 c oramine- o orm pin via e co ore ru azoic aci •
The reaction forms the basis of a spectrophotometric procedure
for ammonia determination. Prochazkova 108 evaluated
65
critic~lly the conditions of the reaction and proposed
the mechanism of reaction. ferrous ion, sulfate ion,
cyanide ion, thiocynate ion, amines and amino acids are
reported to interfer in the procedure.
Ammonia is chlorinated to trichloramine with excess
of hypochlorite solution. The unreacted hypochlorite is
destroyed with nitrite. The absorbance of reaction f
mixture after treatment with cadmium iodide-starch is
measured at 615 nm.109
On mixing with triphenyl phosphine-ruthenium
solution,ammonie forms a complex which is extracted with
benzene. The absorbance of the organic layer is
measured at 620 nm.110
Other spectrophotometric procedures for the
determination of ammonia involve the use of thymol and
chloramine-T,111
thymol and hypochlorite,112
thymol end
113 114 hypobromite, hypobromite, o-benzene-sulfamido-p-
benzoquinone in dioxane115
or mercury (II) and methyl
thymol blue reagent.116
3.1.4 Ammonia Sensing Electrode
In potentiometric estimation of ammonia,using gas
sensing electrode based an internal pH probe are commonly
66
11 7 employed. Recently, several ammonium ion electrodes
. 118-120 with better selectivity have been described to
determine ammonia in concentration range of 10-6
to 10-3
mole/litre. The determination should be carried out at
constant temperature and with continuous stirring.121
The gas reacting with water, as well as volatile amines
interfer in the determination • . f
3.1.5 ~~zymatic Methods
In clinical laboratorie~ ammonia in blood is
usually determined by the enzymatic method in which
glutamate dehydrogenase catalyses the condensation of
ammonia with oe.-ketoglutarate with simultaneous oxidation
of the reduced nicotinamide adenine dinucleotide phosphate
(NADPH). The decrease in absorbance at 340 nm is
directly proportional to the ammonia concentration.122
3.1.6 Miscellaneous Methods
The other methods for the determination of ammonia
include atomic absorption spectroscopy,123
molecular
124 125 absorption spectroscopy, ' molecular emission
126 127 128 spectroscopy, ' plasma emission spectroscopy,
polarography, 129 • 130 high pressure liquid chromato-
131 132 133 graphy (HPLC), ion chromatography, ' and
t . 1 34 t h . gasome ric ec niques.
l
67
3.2 .[ield Qf Ammonia Determination
3.2.1 Pharmaceuticals
The aqueous ammonia solution is used in various
pharmaceutical processes as a mild alkalizer. It is
often preferred to the fixed alkalies because of its
volatility. The excess of ammonia is readily detected
by its odour and it can be ea,ily removed by heat.
Ammonium salts commonly used in therapy include
the carbonate, chloride and bromide. The carbonate
liberates ammonia copiously which on inhalation, acts
as a reflux stimulant. The bromide is used as a central
depressant and the chloride as systemic acidifier. The
latter enhances the diuresis of organic mercurials.
Both the chloride and carbonate are the common ingredients
in expectorant preparations. Ammonium acetate and
ammonium citrate are also used in pharmaceutical formula-
tions.
The determination of ammonia or ammonium ion
involves back titration, 92 formal titration93
or titration
of liberated ammonia from ammonium salt with the help
of alkali.92
68
Compounds like urea and saccharine are also
determined as ammonia after their decomposition by
treatment with alkali. 135 The ammonia formed is distilled
off in standard acid solution which is then back titrated
with standard alkali solution.
3.2.2 fertilizers
f Generally, ammonium sulphate, ammonium chloride,
diammonium phosphate, ammonium nitrate or urea are used
as nitrogen fertilizers.
The determination of ammonium nitrogen as such or
in the presence of urea and nitrate requires different
methods. AOAC 136 describes the formal titration method
in case of ammonium nitrate and ammonium sulphate
containing fertilizers. It also recommends a method
involving distillation of ammonia in the presence of
magnesium oxide. Ammonium ion in fertilizers is
determined by indophenol method as free ammonium
ion137 • 138 and in combination with urea and nitrate.139
Ammonia selective membrane electrodes have been
d t d . . . f t · 1 · 1 40 use o etermine ammonia in er i izers.
69
(i) Blood
In modern clinical laboratories, ammonia in blood
is 122 usually determined by a~ enzymatic method. After
its separation by Selingson's modified diffusion technique,
. . d . t t 141t142 ammonia is eterm1ned by Nessler s reagen •
In indophenol method, protein/. and other materials are
precipitated by treatment with ice cold 10% w/v of
trichloroacetic acid solution in 1.3% w/v sodium hydroxide
1 t. 143
so u ion.
144 reagent.
(ii) Urine
Serum ammonia is also .estimated by Nessler' s
Various nitrogen containing compounds are present
in urine. They are in form of inorganic salts of
ammonia as well as the organic nitrogenous compounds.
Ammonia and urea are excreted in major proportion in
urine.
For colorimetric analysis, ammonia is separated
by microdiffusion or distillation prior to the color
development in order to minimise the interference by
ether nitrogenous substances. Most widely used
145 procedures are Nessler's method, and indophenol
th d 146, 14 7 me o •
70
The ammonia formed by treatment of blood serum or
urine with the enzyme (urease) is determined by
Nessler's reagent. The color intensity of the reaction
148 mixture is measured at 420 nm. It is also determined
. . d h 1 t• 149,150 using in op eno reac 10~.
3.2.4 Miscellaneous
Food materials like milk~· fish, meat, etc~ contain
free ammonia. The concentration of ammonia increases on
decomposition of fish and meat. For checking their
quality, it is necessary to determine the free ammonia
content of these food materials. Free ammonia is also
present in water, and air. In all these cases, the
colorimetric procedure of Nessler's reagent and indophenol
method are employed to determine ammonia.
~termination of Ammonia and Ammonium Ion Using H~~£~
Reaction
Hantzsch reaction is mainly employed in the synthesis
cf pyridines. When ammonia reacts with acetylacetone and
formaldehyde in aqueous media, yellow colored compoundJ
3,5-diacetyl-1 ,4-dihydrolutidin~ is produced. Belman 17
used this reaction for the detection of ammonia. However,
the reaction is not investigated in details for the
71
quantitative determination of ammonia. It was, therefore,
thought of interest to study the reaction conditions of
Hantzsch reaction and to develop a new colorimetric
method for determination of ammonia.
In the present work, various reaction conditions
such as concentration of reactants, pH, time and
temperature of reaction, etc.fare standardized to obtain
maximum color intensity. The method is based on formation
of 3,5-diacetyl-1,4-dihydrolutidine under the reaction
conditions.
Pure samples of ammonia, its salts and pharmaceutical
formulations are analysed by proposed method. The method
is applied to the determination of ammonia nitrogen in
fertilizers. The results compare favourably with those
obtained by official method.
72
3.3.1 ~parat~
As described in Chapter 2.
Ammonium Sulphate (BDH), Ammonium Chloride (BDH),
Ammonium Bicarbonate (BDH), D~ammonium Phosphate (SD'S), f"
Ammonium Nitrate (BDH), Ammonium Acetate (BDH),
Urea (BDH), Potassium Hydrogen Phthalate AR (BDH),
Potassium Dihydrogen Phosphate AR (BDH), Potassium
Chloride AR (BDH), Sodium Acetate (anhydrous) (BDH),
Cupric Chloride (BDH), Formaldehyde (BDH),
Hydrochloric Acid (BP), Acetic Acid (BDH), Acetyl
ecetone (SD'S), Sodium Hydroxide (Pellets) (BDH),
double distilled water were used·~n the study.
3.3.2.1 f..!:eparation of Buffer Solution
The buffer solutions of pH 3.0 to 6.0 were prepared
by mixing potassium hydrogen phthalate solution (D.2M)
and hydrochloric acid solution (D.2M) or sodium hydroxide
81 solution (D.2M).
The buffer solution of pH 3.45 to 5.B were
prepar~d by mixing sodium acetate solution (D.2M) and
acetic acid solution (D.2M).151
73
The final pH of buffer solutions was adjusted
on pH meter.
The reagent solution was prepared by mixing formal- •
dehyde (150 ml) and freshly distilled acetylacetone (78 ml)
with distilled water.
1 litre with water.
The final volume was adjusted to
f
3.3.2.3 Preearation of Bufferred R!a~ent Solutii
The reagent solution was prepared by mixing
acetate (16.4 g}, acetic acid (12 ml), formaldeh~
{150 ml) and freshly distilled acetylacetone (78 rr,,
The volume was adjusted to 1 litre with distillec
Tbe final pH of the solution was 4.7 ~ 0.1.
3.3.2.4 Preparation of Standard Ammonium Sulphate
Solution
Ammonium sulphate (472.D mg) {previously dried at
105°c for 2 hours) was dissolved in and diluted to 100 ml
with water. The solutio~ (5 ml) was diluted further
to 100 ml with the same solvent. Final solution
contained 50 mcg of nitrogen per ml of the solution.
74
3.3.2.5 PreEa~a!~on of Standard Ammonium Chloride
Solution
Ammonium chloride (382.0 mg) was dissolved in end
diluted to 100 ml with water. The solution (5 ml) was
diluted further to 100 ml with the same solvent.
3.3.2.6 ~~~earation of Stand~~_!!iammonium Pho~e~
Solution
Accurately weighed diammonium phosphate (471 .4 mg)
was dissolved in and diluted to 100 ml with water. The
solution (5 ml) was diluted further to 100 ml with water.
3.3.2.7 PreEara.ii.£n of Standard Ammonium Nitrate
Solution
Previously dried ammonium nitrate (571.4 mg) was
dissolved in and diluted to 100 ml with water. The
solution (5 ml) was diluted further to 100 ml with the
same solvent.
3.3.3 Procedure
absorbance
Standard ammonium sulphate solution (2.D ml) was
pipetted into a 100-ml conical flask. Bufferred reagent
75
solution (4 ml) was added to it. The reaction mixture
was immersed in a boiling water bath for 15 minutes.
It was cooled to. room temperature. The content of
flask was transferred quantitatively into a 25-ml
volumetric flask' with the help of water. The volume
was adjusted to the mark with water. The absorbance
of the colored solution was measured on Beckman Model 25 f
spectrophotometer in the range of 350 to 550 nm against
blank.
The blank was prepared similarly in which volume
of standard ammonium sulphate solution was replaced
by equal volume of water.
Maximum absorbance of the colored solution was
obtained at 412 nm (Fig.16).
76
Standard ammonium sulphate solution (0.25, 0.5, 1.0,
1 .5, 2.0, 3.0 and 4.0 ml) was pipetted into a series of
100-ml conical flasks. Sufficient water was added to .
each flask to obtain 4ml of reaction mixture. The bufferred
reagent solution (4 ml) was added to it. The flasks were
heated for 15 minutes in boiling water bath. After cooling f'
to room temperature, the contents of the flask were
transferred quantitatively to 25-ml volumetric flasks with
the help of water and diluted to the mark with water.
The absorbance of the colored product was measured at
412 nm against blank (Fig.17). The blank was prepared by
replacing standard ammonium sulphate solution with equal
volume of water.
3.3.3.3 ~ert-Be~~f~~_f2.E, Ammonium Chloride,
Standard solutions of ammonium chloride, diammonium
phosphate or ammonium nitrate (0.25, 0.5, 1.0, 1 .s, 2.0,
3.0 and 4.0 ml) were pipetted in to 100-ml conical flasks
and analyzed as described under 3.3.3.2 (Fig.18,19,20).
I~
77
3.3.4 [actors affecting the deve~opment of color
Standard ammonium sulphate solution (2.D ml) was
treated with buffer solutions (phthalate or acetate buffer)
of varying pH (3.D to 6.0; 4 ml) and the non-bufferred
reagent (4 ml). The reaction mixture was immersed in a
boiling water bath for 15 min~tes. ~ The mixture was
cooled to room temperature and transferred quantitatively
to a 25-ml volumetric flask. It was diluted to the mark
with water. The absorbance of the colored solution was
measured at 412 nm against blank prepared by replacing
ammonium sulphate solution with equal volume of water.
Maximum absorbance was observed at pH 4.4 to 5.0
(Fig.21).
3.3.4.2 Effect of acetate buffer concentration -----Different volumes of the acetate buffer solution
(pH 4.7; 1 to 7 ml) were pipetted into a series of 100-ml
conical flasks containing standard ammonium sulphate
solution (2.D ml). Non-bufferred reagent solution (4 ml)
was added to each f lesk and analyzed as described under
3.3.3.1. The ebsorbance was measured at 412 nm against
blank.
78
Maximum absorbance was observed in presence of
3 ml buffer solution which remained constant on increasing
buffer concentration (Fig.22). In the present work, 4 ml
buffer solution was used.
The absorbance at 412 nm of the colored products
formed by the reaction of steridard ammonium sulphate
solution (2.0 ml} and buffer solution (pH 4.7; 4 ml) with
different volume of non-bufferred reagent solution
increased with the increase in the concentration of the
non-bufferred reagent solution (fig.23).
Maximum color intensity was obtained in the presence
of 4 ml of the reagent solution which remained constant
with further increase in the volume of reagent solution.
3.3.4.4 ~ct of Temperature
The reaction of standard ammonium sulphate solution
(2.0 ml) in presence of acetate buffer (pH 4.7; 4 ml) with
non-bufferred reagent (4 ml) was carried out at different
0 0 0 0 . . temperatures (25 , 30 , 53 and 100 C) for 30 minutes.
Absorbance of the reaction mixtures was measured at
412 nm (fig.24).
Maximum color intensity was obtained et 100°c.
79
3.3.4.5 Effect of time of reaction
The reaction of standard ammonium sulphate solution
(2.0 ml) with non-bufferred reagent solution (4 ml) in the
presence of acetate buffer (pH 4.7; 4 ml) was carried out
in boiling water bath for different time intervals and
analyzed as described under 3.3.3.1. Absorbance of the
reaction mixture was measured ft 412 nm against blank.
Maximum color intensity was obtained after 15 minutes
(Fig.25) which remained constant on further heating.
Standard ammonium sulphate solution (2.D ml) was
reacted as described under 3.3.3.1 to form a yellow color
product. One set of reaction mixture was stored in dark
in stoppered bottle and another set was allowed to stand
in diffuse sunlight. After different time intervals
of storage~the color intensity was measured at 412 nm
(Fig.26).
The absorbence of the reaction mixture stored in
dark was constant for more then 24 hours, while that of
the reaction mixture kept in diffuse sunlight was constant
upto 4 hours.
80
3.9.5.1 Synt~~of 3,5-diacetyl-1,4-dihydrolutidine
Ammonium acetate (7.7g; 0.1M) was dissolved in water
(200 ml) in a round bottom flask (500 ml). Acetylacetone
(20g; 0.2M) and formaldehyde (7.Sml; 40%) were added to it.
The reaction mixture was boiled immediately for 5 minutes
with continuous stirring. It was allowed to cool to room
temperature. The yellow soli4·separated was filtered.
The residue was washed with water and dried. On ~ecrysta
o llization from ethanol,the product melted at 196-199 c.
{reported12 M.P. 19B
0 c).
~lys~
Molecular formula
3 Calculated for N
3 found : 7.24
3.3.6.1 ~~alysis of Ammonium Sulphate
The powdered ammonium sulphate (ca.472mg) was
weighed accurately and dissolved in water and diluted to
100 ml with the same solvent. The solution (5 ml) was
diluted further to 100 ml with water and analysed as
described under 3.3.3.1 (Table IV).
81
3.3.6.2 Analysis of Ammonium Chloride
Ammonium chloride (ca.382.0 mg) was weighed accurately end
dissolved in and diluted to 100 ml with water. The solution
(5 ml) was transferred to 100-ml volumetric flask and '
diluted upto the mark with same solvent. Aliquot (2.0 ml)
of the solution was analyzed ~s described under J.3.3.1.
The amount of ammonium chlori~~ was determined by referring
to the standard curve (Table vi.
3.3.6.3 Anal~sis of Ammonium Bicarbonate
Ammonium bicarbonate (ca.S64.7mg) was dissolved in
and diluted to 100 ml with water. The solution (5 ml)
was diluted further to 100 ml with the same solvent and
analysis was carried out as described under 3.3.3.1
(Table V).
3.3.6.4 Analysi~f Diammonium EJ!osehate
The powdered diammonium phosphate (ca.472 mg) was
weighed accurately and dissolved in and diluted to 100ml
with water. The solution (5 ml) was diluted further to
100 ml with water and analyzed as described under 3.3.3.1
(Table V).
82
3.3.6.5 Anal~sis of Ammonium Nitr~!
Ammonium nitrate (ca.571 .4mg)was weighed accurately
and dissolved in and diluted to 100 ml with water. The .
solution (5 ml) was diluted further to 100 ml with the
same solvent and analyzed as described under 3.3.3.1
(Table V).
3.3.6.6 Analysis~Ammonium Acetate
Ammonium acetate (ca.550.6mg)was weighed accurately
and dissolved in and diluted to 100 ml with water. The
solution (5 ml) was diluted further to 100 ml with same
solvent and its analysis was carried out as described
under 3.3.3.1 (Table V).
3.3.6.7 Ana!~sis of Strone Solution of Ammonia
The strong solution of ammonia (ca.~00 mg) was
weighed accurately into a 100-ml volumetric flask
containing 10 ml of water and diluted to mark with
water. The solution (5 ml) wes further diluted to
100 ml with the same solvent. The dilute solution (~Oml)
was analyzed as described under 3.3.3.1 (Table VI).
I~
83
3.3.6.8 Analysis of Dilute Solution of Ammonia
The dilute solution of ammonia (ca.1.2 g) was weighed
accurately and dissolved in and diluted to 100 ml with
water. The solution (5 ml) was further diluted to 100 ml
with the same solvent and the dilute solution (~Oml) was
analyzed as described under 3.3.3.1 (Table VI). f'
3.3.6.9 Analysis of Strong Solution of Ammonium Acetate
The strong solution of ammonium acetate (0.1 ml) was
diluted to 100 ml with water. The solution (5 ml) was
diluted further to 100 ml and the dilute solution (ZOml)
was analyzed as described under 3.3.3.1 (Table VI).
3.3.6.10 Analysis of Ichthammol
Ichthammol (ca.300 mg) was weighed and dissolved
in water (5 ml). Copper chloride solution (2%; 2 ml) was
added to it with constant stirring, and was kept aside
for 20 minutes. The mixture was filtered through Whatman
filter and washed thoroughly with water. The filtrate
and washing were combined and diluted to 100 ml with the
same solvent. The solution (2.0 ml) was analyzed as
described under 3.3.3.1 (Table VI).
84
3.3.6.11 Anel~sis_of ammonium ni!Eoaen ~the eresence
urea in s~nthetic mixture
The synthetic mixtures of diammonium phosphate and
urea were prepared (Table VII). The mixture equivalent
to ammonium nitrogen (100 mg) was weighed accurately end
dissolve in water end diluted to 100 ml. An aliquot
(5 ml) of the solution was diluted further to 100 ml with
water and the solution (2.0 ml) was analyzed es described
under 3.3.3.1 (Table VII).
85
3.4 Results and Discussion
Ammonia has been determined spectrophotometrically
using various methods. In most of the methods, the reaction
is carried out in alkaline media. Highly alkaline medium
causes loss of ammonia from the solution if the reagents
are not added immediately. They are time bound and required
fix order of r~agent additionf~ith many steps. The presence
of some other ions interfere in the analysis. Therefore,
it was desirable to establish a simple, accurate and precise
method for determination of ammonia as well as ammonium ion.
Belman19 detected arnmonie fluorimetrically using
Hantzsch reaction. In the present work, the reaction
conditions are standardized to estimate ammonia colori-
metrically.
In the proposed method, ammonia was reacted with
acetylacetone - formaldehyde reagent (4 ml) prepared in
acetate buffer (pH 4.7~ in boiling water bath. The yellow
colored product formed showed maximum absorbance et 412 nm
(Fig.16). The maximum color intensity was obtained after
keeping the reaction mixture in boiling water bath for
15 minutes (Fig.25). The color remains stable for 4 hr
86
in diffuse day light and for more than 24 hours, if
stored in a stoppered bottle in dark (fig.26). Maximum
color intensity was observed when the reaction carried
out in a buffer of pH 4.~ to 5.0 (Fig.21). The Lambert-
Beer's law is obeyed in the concentration range of D.5
to B mcg of ammonium nitrogen per ml. of the reaction
mixture (Fig.17). The optimup concentration range for
the determination as evaluated from the Ringbom plot83
was found to be 1 to 6 ppm of ammonia nitrogen. The
effective molar absorptivity in terms of ammonia was
-3 -1 -1 3 -1 -1 found to be 1 • 7 x 1 0 L mol cm or 1 .4 x 10 L mol cm
in terms of ammonia nitrogen and the photometric
•t• •t 84 f th 1 t• f d t b 0 01 sensi 1v1 y o e co or reac ion was oun o e • mcg
-2 of ammonia nitrogen cm at 412 nm. The standard and
percentage relative standard deviations were found to be
!_D.713 and D.715%_,respectively (Table IV). These
results indicate that the method is precise and gives
reproducible results.
Pure samples of pharmaceutical ammonium salts and
its official formulations, as well as Strong and Dilute
Solution of Ammonia were analysed by the proposed method
and compared with official method 136
•152
(Table V,VI).
The results by both the methods are in good agreement.
I~
87
137 There is no approved AOAC procedure for the
determination of ammonical nitrogen in urea-ammonium
phosphate based fertilizers. In the preliminary experiment,
it was found that urea does not interfer in the determine-
tion of ammonia nitrogen by proposed procedure •. Various
synthetic mixtures containing urea with diammonium
phosphate were analyzed by the proposed method. The
satisfactory recovery of DAP '~s obtained (Table VII).
lchthammol contains ammonia in form of sulphate as
well as in form of sulphonate. Pharmacopoeial ichthammol
is assayed directly by the proposed procedure after the
precipitation of resinous matter with copper chloride
solution. However, the commercial samples do not give
clear solution after precipitation and hence they could
not be analyzed by the proposed procedure. They are
l d t . f t by USP 153 d ana yze sa is ac ory proce ure.
In order to study the nature of the chromophore,
3,5-diacetyl-1,4-dihydrolutidine was synthesized by
reacting ammonium acetate with acetylacetone and formal-
dehyde in aqueous medium. The product was dissolved in
aqueous ethanol and scanned in the range of 350 to 550 nm
(Fig.27). The reaction mixture containing ammonium
I
~
88
sulphate solution (Zoml) and buffered reagent solution
(4 ml) was also scanned between 350 to 550 nm. It shows
similar characteristic of 3,5-diacetyl-1,4-dihydrolutidine
with wave length of maximum absorbance at 412 nm (fig.27}.
It is evident from the above observation that
3,5-diacetyl-1,4-dihydrolutidine formed in the reaction
mixture is responsible fof the color.
1~
Q)
u c: co ..c
J..t
o.6
0.5
0.4
89
0 0 .3 (I)
..0 ct:
0.2
0 .1
350 400 450 500 550
Wavelength (nm)
Figure 16 : Visible spectrum of the colored product
obtained on reacting Ammonium Sulphate
with reagent.
90
0.9
o.a I
It 0.1
E o.6 c
(\J
~
+' IO o.s Cll u c co .0 H D.4
~· 0 Ill
I .0 <!
0.3
0.2
0 .1
0 1 2 3 4 5 6 7 8
Ammonium N {mcg/ml)
Figure 17 : Lambert-Beer's curve for Ammonium Sulphate.
91
D.9
o.s
0.1
o.6
E c
N o.s .-- '• 'l:t
+> m
QJ D.4 fU 1C ro
.D H a Ill .D er: 0.3
0.2
0 .1
0 1 2 3 4 5 6 7 8
Ammonium N {mcg/ml)
Figure 18 : Lambert-Beer's curve for Ammonium Chloride.
92
o.a
t 0.1
o.6
o.s
D.4
o.3
0.2
0. 1
0 1 2 3 4 5 6 7 8
Ammonium N (mcg/ml)
Figure 19 Lambert-Beer's curve for Diammanium Phosphate
93
0.9
O.B
0.1
o.6 E c:
N .... o;t
+> o.s
ID
QJ u
IC tU
0.4 .D H a 0)
.0 ~
0.3
0.2
0. 1
0 1 2 3 4 5 6 7 8
Ammonium N (mcg/ml)
Figure 20 : Lambert-Beer's curve for Ammonium Nitrate.
94
I~
- 0 - o-e-o-o- Buffer8 1
Acetate buffer
o.s
E c D.4
('\J
o' '<;t I
+> I
m 0.3 I
0
Q.J I
u I c I -\ IU 0 'o .0 I H i 0 0.2 I (I)
" ..a ~
? I
0. 1 QI
I
I
'
( 0' 2) 3 4 5 6 8
pH
Figure 21 Effect of pH on color intensity.
I
I~
E c
N
v ~
ID
m u c ID .0 H 0 !I)
.0 <t:
o.6
o.s
0.4
D.3
0.2
0 .1
0 1
Figure 22
95
2 3 4 5 6
Buffer (in ml)
Effect of buffer (pH 4.7} concentration on
color intensity.
96
o.6
o.s
E c
N D.4 .-<::l'
+> ro QJ
u D.3 c: Ill .0
1-1 0 (I) .0 <t: 0.2
0 .1
0 1 2 3 4 5 6 1
Reagent (in ml)
Figure 23 : Effect of reagent concentration on color
intensity
I
~
e c
N
~
+> ID
Q)
u c IC
..0 H 0 (I)
.D <t:
o.6
o.s
0.4
D.3
0.2
0 .1
0 20
Figure 24
40
97
60 80
0 Temperature ( C)
1 00
Effect of temperature on color intensity.
98
I
~
o.s
E 0.4 c N .-
""" ..µ
0.3 m
Ill u c m .a ~ 0.2 0 (/)
.a <:
0 .1
0 5 1 0 1 5 20 25 30
Time (in minutes)
Figure 25 Effect of heating time on color intensity.
99
I
r\
o.6
in diffuse sunlight
o.s in dark
E c
N D.4 - )I .....
..-- ~ ..... '<;t
)( ...... ~
...,. Ill
QJ o.3 u c ro
.D 1-1 0 II) 0.2 ..0
<C
0. 1
0 2 4 6 8 24
Time (in hours)
Figure 26 : Effect of time on stability of colore~ product.
0.5
D.4
Ill u o.3 c: ltl .0
J..f 0 II)
..0 <
0.2
0 .1
350
Figure 27
400
100
450
Ammonium Sulphate
3,5-Diacetyl-1,4-dihydrolutidine
500 550
Wavelength (nm)
Comparision of visible spectrum of colored
product obtained an racting Ammonium Sulphate
with reagent, with 3,5-diacetyl-1 ,4-
dihydrolutidine.
----~"
~
1 01
TABLE IV
fu!!~sis of Ammonium Nitro~en
No. % N Recover~ b~
Proposed method Official method1 36
1 1 01 • 00 f 101.40
2 100.00 99.20
3 99.00 98.60
4 99.60 99.60
5 98.60 99.80
6 100.20 1 01 .oo
1 100.40 100.20
B 99.40 100.60
9 99.80 99.60
10 1 00 .20 99.00
AV 99.82 99.90
SD ± o. 713 + 0.896 -RSD :t: 0. 715 % + 0.697 % -
1 02
i\
TABLE V
Analysis of Ammo/l.ium Sal ts
Name of compound % Recover~ .:t SD a) b~
Proposed Official method method136,152 -- -----
1 • Ammonium Sulphate 99.98 + 0.10 99.99 + D.95 -2. Ammonium Chloride 100.00 + 0. 71 99.91 + O.BB - -3. Ammonium Bicarbonate 1 00. 78 + 0.98 99.98 + D.99 - -4. Ammonium Nitrate 99.93 + D.99 99.95 + 1 .1 0 -s. Diammonium Phosphate 1OD.52 + ·o.1s 1 OD .1 2 + o.eo - -6. Ammonium Acetate 96.98 + 1 .03 96.80 + 1 • 21 - -
a) Standard deviation was calculated from the results
of nine determinations.
103
TABLE VI
Analysis of Ammonia and its formulations
No.
1 •
2.
3.
Name of compound.
Strong solution of Ammonia
Dilute Solution of Ammonia
Strong Solution of Ammonium Acetate
4. Ammonium sulphate in Ichthammol
(a) Pharmacopoeia!
(b) Commercial-A
(c) Commercial-B
% found a) . by Reference
Prc;y>osed Official me~hod method
28.B2w/w 2B.57w/w 152
9 .84 w/w 9 .69 w/w 152
5B.BOw/v se.o5w/v 152
12.59 w/w 12.SB w/w 153
13.S4w/w 153
14.0Sw/w 153
---------a) Average value of five determinations.
104
TABLE VII
Ana~~sis of Ammonium ~itrogen.in Diammonium phosphat~ 1
in presence of urea in s~nthetic mixture
Diammonium Urea 'Jb Recovery a)of ammonium phosphate (in mg) nitrogen by proposed (in mg)
method --1 472 50 100.40
2 480 100 1 00 .15
3 470 200 1 00 .42
4 460 300 1 DO .25
a) Average value of five determinations.